This is a request for an ADAMHA SDA grant. The overall goal of this project is to elucidate the molecular mechanism of the cellular trafficking of human muscarinic cholinergic receptor, Hm 1. Deficits in the cholinergic system in the central nervous system may contribute to memory impairments associated with neurodegenerative disorders such as Alzheimers disease, and cholinergic therapy has shown some limited success in treating these disorders. For the success of any long-term therapy, receptor regulation is an essential factor involving desensitization and downregulation. The molecular and cellular mechanisms of downregulation remain unknown at present for any G protein coupled receptor. This project focuses on the processes of internalization, sequestration, recycling, and downregulation of Hm 1. The initial goal is to identify the receptor domains involved in controlling these processes in Hm 1 receptor, and preliminary results suggest that more than one region of the receptor may be involved in the process of internalization. The novel hypothesis will be tested that receptor domains exist that inhibit, rather than mediate, receptor internalization. If correct, such domains could account for the persistence of many G protein coupled receptors at the cell surface even after agonist-activation, and for receptor sequestration without internalization. The second part of the project will involve studying the cellular mechanisms of receptor internalization. The role of phosphorylation in receptor sequestration, internalization, recycling and downregulation will be elucidated with the use of point mutations at serine and threonine residues in the third cytoplasmic loop and carboxy tail of the receptor. The cellular pathway, clathrin coated pits or noncoated vesicles, involved in internalization of the Hm 1 receptor will be determined. In addition the major proteins mediating these cellular processes will be identified. Finally the functional state of the internalized Hm 1 receptor, with respect to maintaining its coupling to a specific G protein, will be revealed. These combined results are expected to enhance significantly the current understanding of the regulation of a prototype G protein coupled receptor. This enhanced knowledge can be further extrapolated to other G protein coupled receptors which include many neurotransmitter and hormonal receptors.